Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/07—Nucleotidyltransferases (2.7.7)
  • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

• the present disclosure relates to a phi29 DNA polymerase mutant with improved thermal stability and application thereof.
• Phi29 DNA polymerase belonging to the family B DNA polymerase, is a DNA polymerase derived from Bacillus subtilis phi29 phage.
• the crystal structure of phi29 DNA polymerase shows that phi29 DNA polymerase has two unique domains, i.e. TPR1 and TPR2 domains, in addition to conserved domains Palm, Thumb, Finger and Exo which are contained in common family B DNA polymerases, in which such a TPR2 domain takes part in forming a narrow channel surrounding the downstream DNA strand template, making the double-stranded DNA dissociated; meanwhile, the Palm, Thumb, TPR1 and TPR2 domains constitute a circular structure which tightly binds to the upstream double strands newly formed by template strand.
• the phi29 DNA polymerase Due to its structural characteristics, the phi29 DNA polymerase has a specific high processivity, strong strand displacement activity and 3' ⁇ 5' exonuclease correction activity, thus commonly used in thermostatic amplification process, such as Rolling Circle Amplification (RCA) of micro amount of circular plasmids, Multiple Displacement Amplification (MDA) of genome and the like, and further applied to steps of library preparation through high-throughput sequencing, strand displacement amplification and the like.
• RCA Rolling Circle Amplification
• MDA Multiple Displacement Amplification
• Phi29 DNA polymerase is a mesophile enzyme, with a poor thermal stability, which can be inactivated by heating at 65°C for 10 minutes. In practice, the storage life of phi29 DNA polymerase product, effect of DNA amplification and sequencing and the like are ofen affected due to the poor thermal stability.
• the current research mainly focuses on aspects of 1) optimization of storage buffer or reaction buffer, such as adding some surfactants or compatible solutes, and 2) mutating the protein sequence of wild-type phi29 DNA polymerase or constructing a chimeric protein.
• the object of the present disclosure is to provide a phi29 DNA polymerase mutant with improved thermal stability and application thereof.
• the present disclosure in embodiments provides a protein, which is obtained by subjecting phi29 DNA polymerase to point mutation A and/or point mutation B and/or point mutation C, wherein the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to other amino acid residues; the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to other amino acid residues; and the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to other amino acid residues.
• M Methionine
• L amino acid residue Leucine
• E amino acid residue Glutamic acid
• the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to Histidine (H), Alanine (A) or Lysine (K);
• the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to Lysine (K), Phenylalanine (F), Isoleucine (I) or Histidine (H);
• the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to Glycine (G) or Proline (P).
• the protein is any one selected from protein (1) to (19):
• the protein may be a fusion protein obtained by ligating a His 6 tag at terminus of any protein of (1) to (18). More specifically, the protein may be a fusion protein obtained by ligating a His 6 tag at the N-terminus of any protein of (1) to (18).
• the protein as described above has increased stability compared to the phi29 DNA polymerase.
• the stability is thermal stability. More specifically, the thermal stability may be a thermal stability at 37°C.
• the present disclosure in embodiments also provides a nucleic acid molecule encoding the protein as described above, an expression cassette containing the nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, a recombinant bacterium containing the nucleic acid molecule, and a transgenic cell line containing the nucleic acid molecule.
• the nucleic acid molecule may be any DNA molecule of (1) to (19):
• the recombinant vector is obtained by inserting the nucleic acid molecule into an expression vector.
• the expression vector may be a pET28a (+) vector.
• the recombinant bacterium is a bacterium obtained by introducing the recombinant vector into an original bacterium.
• the original bacterium may be Escherichia coli (E. coli).
• the Escherichia coli may be E. coli BL21 (DE3).
• the transgenic cell line may be obtained by transforming the recombinant vector into recipient cells.
• the transgenic cell line is a non-plant propagative material.
• the present disclosure in embodiments further provides use of the protein as described above for any one of (a) to (g):
• the present disclosure in embodiments further provides use of the nucleic acid molecule encoding the protein as described above, the expression cassette containing the nucleic acid molecule, the recombinant vector containing the nucleic acid molecule, the recombinant bacterium containing the nucleic acid molecule and the transgenic cell line containing the nucleic acid molecule, for any one of (h) to (k):
• the present disclosure in embodiments further provides a method of improving the stability of phi29 DNA polymerase, comprising subjecting phi29 DNA polymerase to point mutation A and/or point mutation B and/or point mutation C, wherein the point mutation A is the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to other amino acid residues; the point mutation B is the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to other amino acid residues; and the point mutation C is the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to other amino acid residues.
• M Methionine
• L amino acid residue Leucine
• E amino acid residue Glutamic acid
• the point mutation A may be the mutation of amino acid residue Methionine (M) at position 97 of the phi29 DNA polymerase to Histidine (H), Alanine (A) or Lysine (K);
• the point mutation B may be the mutation of amino acid residue Leucine (L) at position 123 of the phi29 DNA polymerase to Lysine (K), Phenylalanine (F), Isoleucine (I) or Histidine (H);
• the point mutation C may be the mutation of amino acid residue Glutamic acid (E) at position 515 of the phi29 DNA polymerase to Glycine (G) or Proline (P).
• the method as described above may be any one of procedures (1) to (18):
• the stability may be thermal stability. More specifically, the thermal stability may be a thermal stability at 37°C.
• the phi29 DNA polymerase may be (I), (II) or (III):
• pET28a (+) vector is from Novagen.
• E. coli BL21 (DE3) is from TIANGEN, in a catalog number of CB105-02.
• Storage buffer includes 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5%(v/v) Tween® 20, 0.5% (v/v) NP-40 and 50% (v/v) Glycerol, with pH7.4 @ 25°C.
• 141 RCA Primer in the examples is of a sequence: TCCTAAGACCGCTTGGCCTCCGACT.
• 141Ad ssDNA in the examples is generated by BGI and is a circular single-strand library in a certain size range, without fixed sequences. Specifically, it is a random library consisting of four nucleotides (A/T/C/G), with a main band in a length of 200 to 300 bp.
• a wild-type recombinant vector (recombinant vector WT) was obtained by inserting the DNA molecule as shown in SEQ ID NO: 2 in the sequence listing between the Nde I and BamH I restriction sites of pET28a (+) vector.
• the DNA molecule as shown in SEQ ID NO: 2 in the sequence listing expresses the protein as shown in SEQ ID NO: 1 in the sequence listing, that is, the wild-type phi29 DNA polymerase represented by WT.
• the recombinant vector M97A differs with the recombinant vector WT only in that the nucleotides 289-291 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "ATG” to "GCG", with mutated DNA molecule encoding mutant M97A.
• the mutant M97A differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A).
• the recombinant vector M97K differs with the recombinant vector WT only in that the nucleotides 289-291 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "ATG” to "AAA", with mutated DNA molecule encoding mutant M97K.
• the mutant M97K differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K).
• the recombinant vector L123K differs with the recombinant vector WT only in that the nucleotides 367-369 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "CTG” to "AAA", with mutated DNA molecule encoding mutant L123K.
• the mutant L123K differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 123 is mutated from Leucine (L) to Lysine (K).
• the recombinant vector E515G differs with the recombinant vector WT only in that the nucleotides 1543-1545 of the DNA molecule shown in SEQ ID NO: 2 in the sequence listing are mutated from "GAA” to "GGC", with mutated DNA molecule encoding mutant E515G.
• the mutant E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97A-L123I differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "GCG” and the nucleotides 367-369 are mutated from "CTG” to "ATT” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123I.
• the mutant M97A-L123I differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A) and the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I).
• the recombinant vector M97A-L123H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to “GCG", the nucleotides 367-369 are mutated from “CTG” to "CAT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123H-E515G.
• the mutant M97A-L123H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97A-L123F-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to “GCG", the nucleotides 367-369 are mutated from “CTG” to “TTT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123F-E515G.
• the mutant M97A-L123F-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A), the amino acid residue at position 123 is mutated from Leucine (L) to Phenylalanine (F) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97A-L123H-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "GCG", the nucleotides 367-369 are mutated from “CTG” to "CAT” and the nucleotides 1543-1545 are mutated from "GAA” to "CCG” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97A-L123H-E515P.
• the mutant M97A-L123H-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Alanine (A), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).
• the recombinant vector M97K-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-E515G.
• the mutant M97K-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97K-L123K-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA", the nucleotides 367-369 are mutated from “CTG” to “AAA” and the nucleotides 1543-1545 are mutated from "GAA” to "CCG” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123K-E515P.
• the mutant M97K-L123K-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Lysine (K) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).
• the recombinant vector M97K-L123F-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA", the nucleotides 367-369 are mutated from “CTG” to “TTT” and the nucleotides 1543-1545 are mutated from "GAA” to "CCG” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123F-E515P.
• the mutant M97K-L123F-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Phenylalanine (F) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).
• the recombinant vector M97K-L123I-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA", the nucleotides 367-369 are mutated from “CTG” to "ATT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123I-E515G.
• the mutant M97K-L123I-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97K-L123H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA", the nucleotides 367-369 are mutated from “CTG” to "CAT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123H-E515G.
• the mutant M97K-L123H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97K-L123I-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA", the nucleotides 367-369 are mutated from “CTG” to "ATT” and the nucleotides 1543-1545 are mutated from "GAA” to "CCG” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123I-E515P.
• the mutant M97K-L123I-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).
• the recombinant vector M97K-L123H-E515P differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "AAA", the nucleotides 367-369 are mutated from “CTG” to "CAT” and the nucleotides 1543-1545 are mutated from "GAA” to "CCG” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97K-L123H-E515P.
• the mutant M97K-L123H-E515P differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Lysine (K), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Proline (P).
• the recombinant vector M97H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to "CAT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97H-E515G.
• the mutant M97H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97H-L123I-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to “CAT", the nucleotides 367-369 are mutated from “CTG” to “ATT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97H-L123I-E515G.
• the mutant M97H-L123I-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Histidine (H), the amino acid residue at position 123 is mutated from Leucine (L) to Isoleucine (I) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• the recombinant vector M97H-L123H-E515G differs with the recombinant vector WT only in that the nucleotides 289-291 are mutated from “ATG” to “CAT", the nucleotides 367-369 are mutated from “CTG” to “CAT” and the nucleotides 1543-1545 are mutated from "GAA” to "GGC” respective to the DNA molecule shown in SEQ ID NO: 2 in the sequence listing, with mutated DNA molecule encoding mutant M97H-L123H-E515G.
• the mutant M97H-L123H-E515G differs with the wild-type phi29 DNA polymerase only in that the amino acid residue at position 97 is mutated from Methionine (M) to Histidine (H), the amino acid residue at position 123 is mutated from Leucine (L) to Histidine (H) and the amino acid residue at position 515 is mutated from Glutamic acid (E) to Glycine (G).
• Different recombinant bacterium was obtained by introducing respective recombinant vector constructed in step 1.1 into E. coli BL21 (DE3).
• Recombinant bacteria obtained were respectively named as recombinant bacterium WT, recombinant bacterium M97A, recombinant bacterium L123K, recombinant bacterium E515G, recombinant bacterium M97A-L123I, recombinant bacterium M97A-L123H-E515G, recombinant bacterium M97A-L123F-E515G, recombinant bacterium M97A-L123H-E515P, recombinant bacterium M97K-E515G, recombinant bacterium M97K-L123K-E515P, recombinant bacterium M97K-L123F-E515P, recombinant bacterium M97K-L123I-E515G, recombinant bacterium M97K-L123H-E515G, recombinant bacterium M97K-L123I-
• the recombinant bacteria obtained in step 1.2 were respectively subjected to induction and purification, thus obtaining proteins fused to His 6 tag at N-terminus.
• Such proteins obtained were respectively named as a wild-type phi29 DNA polymerase with His 6 tag, a mutant M97A with His 6 tag, a mutant L123K with His 6 tag, a mutant E515G with His 6 tag, a mutant M97A-L123I with His 6 tag, a mutant M97A-L123H-E515G with His 6 tag, a mutant M97A-L123F-E515G with His 6 tag, a mutant M97A-L123H-E515P with His 6 tag, a mutant M97K-E515G with His 6 tag, a mutant M97K-L123K-E515P with His 6 tag, a mutant M97K-L123K-E515P with His 6 tag, a mutant M97K-L123F-E515P with His 6 tag, a mutant
• the recombinant bacteria were inoculated into 3 ml liquid LB medium containing kanamycin, followed by culturing overnight.
• the obtained bacterium solution was transferred into 2 ml liquid LB medium containing kanamycin in volume of 1:100, followed by culturing under shaking at 37°C and 220 rpm to reach an OD 600nm value of 0.6, in which the OD 600nm value in a range of 0.4 to 0.8 is suitable in practice.
• IPTG isopropyl- ⁇ -D-thiogalactoside
• the system was centrifuged at 4°C and 8000 rpm for 5 minutes to collect bacterial cells.
• the bacterial cells obtained in step 1.3.1 were shakly mixed with the suspension buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol; pH 7.9 @ 25°C), ultrasonicated on ice, and centrifuged at 4°C and 12,000 rpm for 30 minutes, thus collecting the supernatant.
• the supernatant obtained in 1.3.2.1 was purified by using nickel column affinity chromatography (HisTrap FF 5ml prepacked column). Specifically, the supernatant was loaded after the column was balanced by 10 column volumes of Buffer A, after which the column was washed with 20 column volumes of Buffer A and eluted with 15 column volumes of eluent consisting of Buffer A and Buffer B, and the eluted solution with target protein was collected. During the elution, the volume fraction of Buffer B increased from 0% to 100% linearly, and the volume fraction of corresponding Buffer A decreased from 100% to 0% linearly.
• Buffer A 20 mM Tris-HCl, 500 mM NaCl, 20 mM Imidazole, 5% (v/v) Glycerol; pH 7.9@25°C.
• Buffer B 20 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, 5% (v/v) Glycerol; pH 7.9@25°C.
• the eluted solution obtained in 1.3.2.2 was purified by using strong anion column chromatography (HiTrap Q HP 5ml prepacked column). Specifically, the eluted solution was loaded after the column was balanced by 10 column volumes of buffer mixture consisting of 59 volume % of Buffer A and 41 volume% of Buffer B. Collection of effluent was started after the protein peak occurred (that is, the UV detection value reached to be 20 mAu), and was stopped until the UV detection value dropped to 50 mAu again.
• Buffer A 20 mM Tris-HCl, 150 mM NaCl, 5% (v/v) Glycerol, pH 7.5@25°C.
• Buffer B 20 mM Tris-HCl, 1 M NaCl, 5% (v/v) Glycerol, pH 7.5@25°C.
• the effluent obtained in 1.3.2.3 was purified by using cation exchange chromatography (HiTrap SP HP prepacked column), thus obtaining a protein sample solution with a purity greater than 95%. Specifically, the effluent was loaded after the column was balanced by 10 column volumes of Buffer A, after which the column was washed with 15 column volumes of Buffer A and eluted with 10 column volumes of eluent consisting of Buffer A and Buffer B. During the elution, the volume fraction of Buffer B increased from 0% to 50% linearly, and the volume fraction of corresponding Buffer A decreased from 100% to 50% linearly. Collection of effluent containing target protein was started after the UV detection value reached to be 50 mAu and was stopped until the UV detection value dropped to 100 mAu again.
• Buffer A 20 mM Tris-HCl, 150 mM NaCl, 5% (v/v) Glycerol, pH 7.5@25°C.
• Buffer B 20 mM Tris-HCl, 1 M NaCl, 5% (v/v) Glycerol, pH 7.5@25°C.
• the target protein obtained in 1.3.2.4 was transferred to a dialysis bag, which was dialysed in the dialysis buffer overnight.
• the protein solution in the dialysis bag was collected and other components were added, thus obtaining a target potein solution containing 1 mg/ml of target potein.
• the other components in the target potein solution are 10 mM of Tris-HCl (pH7.4 @ 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% (v/v) NP-40, 0.5% (v/v) Tween20 and 50% (v/v) Glycerol.
• Dialysis buffer 23.75 mM Tris-HCl (pH 7.4@25°C), 237.5 mM KCl, 2.375 mM DTT, 0.2375 mM EDTA and 5% (v/v) Glycerol.
• the taken target protein solution prepared in Example 1 (as an enzyme solution to be tested) was diluted to 5000 times by volume with the storage buffer, thoroughly mixted by a vortex shaker, and then stilled on ice for 5 minutes to obtain the solution to be tested.
• Pre-reaction system (80.8 ⁇ L): 50 mM Tris-HCl (pH 7.5), 4 mM DTT, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 50 nM dNTP Mixture, 2 pM 141 RCA Primer and 18 ng 141Ad ssDNA.
• the PCR tube was placed on ice when the temperature dropped to 4°C.
• 1 ⁇ l of the solution to be tested was added; and for a negative control group, 1 ⁇ l of storage buffer was added. Both groups were mixed under shaking with a vortex shaker, centrifuged in a centrifuge for 5 seconds and then subjected to a procedure (i.e. heating at 30°C for 60 minutes) in the PCR instrument, with the hot lid set as a temperature of 65°C.
• step 2.2 After the step 2.2, 5 ⁇ l of 0.5M EDTA solution was added to terminate the reaction, and then mixed under shaking.
• ⁇ DNB is the difference of average concentration of reaction products in the reaction-terminated system between the test group and the negative control group, 5000 represents the dilution ratio and 37.38 represents the slope of function between enzyme activity and ⁇ DNB
• the taken target protein solution prepared in Example 1 was divided into two parts, which were respectively treated as follows.
• the target protein solution was placed in a metal bath preheated to 37°C for 10 minutes and centrifuged at 4°C and 13000 rpm for 1 minute to collect the supernatant.
• the supernatant obtained was diluted to 1000 times by volume with the storage buffer, thoroughly mixed by a vortex shaker, and then stilled on ice for 5 minutes to obtain the solution 1 to be tested.
• the target protein solution was diluted to 5000 times by volume with the storage buffer, thoroughly mixed by a vortex shaker, and then stilled on ice for 5 minutes to obtain the solution 2 to be tested.
• the solution 1 to be tested and the solution 2 to be tested were respectively detected according to steps 2.1 to 2.4 in Example 2.
• Example 3 Based on Example 3, several mutants with improved thermal stability were selected and detected for their effect on DNA sequencing through machine test on BGISEQ-500 sequencer according to the standard of BGISEQ-500 sequencer. All reagents used for the test are a complete set of PE50 V2.0 kit produced by BGI, E. coil Ad153 standard library produced by BGI and Qubit ssDNA Assay reagent produced by Invitrogen. The reagents used below are all included in the PE50 V2.0 kit, except for the library and Qubit ssDNA Assay reagent. The PE50 V2.0 reagent tank as described below only refers to the reagents used in the on-machine test.
• DNBs were prepared before on-machine test.
• the DNBs were prepared through the specific steps as below.
• Loading of DNB was conducted according to the specific steps as below: A sample loading reagent plate V2.1 was taken to room temperature for melting, mixed under shaking, briefly centrifuged and placed on ice for use. DNB loading buffer II was taken, shaked for uniformity, briefly centrifuged and placed on ice for use. A chip and the sample loading reagent plate V2.1 were placed in the BGIDL-50. 35 ⁇ l of DNB loading buffer II was added to a PCR tube containing 100 ⁇ l DNB, gently mixed for 15 times with a wide-mouth pipette and arranged in a designated DNB area of the loading system. Loading process was initiated via the DNB loading program (Sample load 2.0), and the loaded chip was incubated at room temperature for 30 minutes and then stored at 2-8°C for use.
• DNB loading program Sample load 2.0
• the protein to be tested was subjected to on-machine sequencing on the BGI SEQ-500 sequencer by using a chip and a BGISEQ-500RS high-throughput sequencing reagent tank (PE50 V2.0).
• sequencing reagent tank II, dNTPs mixture (V3.0) and dNTPs mixture II (V2.0) were thawed and placed in a refrigerator or ice box at 4°C for use; and the DNA polymerase for sequencing was mixed under shaking and placed in an ice box for use.
• a reagent for No. 5 well was formulated, that is, 1150 ⁇ l DNA polymerase mixture and 1150 ⁇ l dNTPs mixture (V3.0) were respectively transferred into the No.
• a reagent for No. 6 well was formulated, that is, 890 ⁇ l DNA polymerase mixture and 890 ⁇ l dNTPs mixture II (V2.0) were respectively transferred into the No. 6 well with a 1 ml pipette, and blew with the pipette for 10 to 15 times for uniformity; and a reagent for No. 14 well was formulated, that is, all reagent for the No. 14 well was taken with a 5 ml pipette, and 2.8 ml of the reagent for the No.
• mutants 97K-123I-515P and 97K-123H-515P are of the lowest value, followed by mutant 97A-123F-515G, and then mutants 97K-123K-515P and 97A-123H-515P, in contrast the wild-type phi29 DNA polymerase exhibits worst performance.
• mutants in the example all have a value of parameter MappingRate% higher than that of wild-type phi29 DNA polymerase.
• the phi29 DNA polymerase in the prior art i.e. wild-type phi29 DNA polymerase
• the present disclosure has screened out several mutants with significantly improved thermal stability from large numbers of phi29 DNA polymerase mutants by using site-directed mutagenesis technology.
• a mutant having a good effect can be further selected by subjecting the amino acids at the mutation sites of the present disclosure to a saturation mutation.
• similar effects can be achieved by mutating other amino acids except for the mutation sites included in the present disclosure.
• mutant protein provided in the present disclosure has significantly improved thermal stability compared to the wild-type protein, which can greatly extend the shelf life of product and effectively improve the sequencing effect of the sequencing platform (such as, BGISEQ-500).
• Such mutant proteins can exist in the form of separately packaged DNA polymerase product or can be packaged in a DNA amplification kit or a DNA sequencing kit.
• the present disclosure also can be used in technical fields of food detection, virus detection, RNA detection, single cell sequencing and the like, as well as development of third or fourth generation sequencers.

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